Quantitative High-Throughput Screening Methods Designed for Identification of Bacterial Biocontrol Strains with Antifungal Properties

ABSTRACT Large screens of bacterial strain collections to identify potential biocontrol agents often are time-consuming and costly and fail to provide quantitative results. In this study, we present two quantitative and high-throughput methods to assess the inhibitory capacity of bacterial biocontrol candidates against fungal phytopathogens. One method measures the inhibitory effect of bacterial culture supernatant components on the fungal growth, while the other accounts for direct interaction between growing bacteria and the fungus by cocultivating the two organisms. The antagonistic supernatant method quantifies the culture components’ antifungal activity by calculating the cumulative impact of supernatant addition relative to the growth of a nontreated fungal control, while the antagonistic cocultivation method identifies the minimal bacterial cell concentration required to inhibit fungal growth by coinoculating fungal spores with bacterial culture dilution series. Thereby, both methods provide quantitative measures of biocontrol efficiency and allow prominent fungal inhibitors to be distinguished from less effective strains. The combination of the two methods sheds light on the types of inhibition mechanisms and provides the basis for further mode-of-action studies. We demonstrate the efficacy of the methods using Bacillus spp. with different levels of antifungal activities as model antagonists and quantify their inhibitory potencies against classic plant pathogens. IMPORTANCE Fungal phytopathogens are responsible for tremendous agricultural losses on an annual basis. While microbial biocontrol agents represent a promising solution to the problem, there is a growing need for high-throughput methods to evaluate and quantify inhibitory properties of new potential biocontrol agents for agricultural application. In this study, we present two high-throughput and quantitative fungal inhibition methods that are suitable for commercial biocontrol screening.

Line 61. What HTP means? Please describe every abbreviature or acronym you use through the text. Furthermore, I think the right abbreviation for high-throughput is "HT". Please revise it. Line 79. Why do you think your proposed method belongs to high-throughput approaches? I do not agree with such statement. To my knowledge HT approaches are designed for testing from hundreds to thousands of samples in a short period of time. Lines 100-101. Spores and mycelium do not have the same structure and composition of their cell walls, so, why not to include mycelium? Figure 1A. Contrasting with the first two plates, I cannot see the fungus nor the bacteria in the third plate. Why? Figure 2A. What should I see in each column? I can see different morphologies and growth, then, I do not know why you said that there is not fungal growth. Please explain. Why did you not include the control of the fungi and the bacteria growing alone? That could help to understand what you meant. I can see fungal growth in all nine wells with different morphology, so why you stated that there is no growth in the first line. Should I see something that you did not explain? Figure 2B. If I apply a zoom on the different wells, I can see growth on the different lines, so I would like to see the control of the fungi and the different Bacilli growing alone under the different conditions. Lines 145-148. I cannot see in figure 2 such dilutions and does not seem to be the initial OD as stated. Have I got confused? Line 152. Did you mean 3.2*10-6. Figure 3A. Above, you concluded that B. amyloliquefaciens and B. velezensis showed the most potent inhibition properties, and even at the highest dilution step against F. culmorum. However, B. subtilis showed similar results. It is necessary an statistical analysis. Lines 191-202. How did you determine the fungal growth by OD In the antagonistic supernatant method? What did you do to avoid clumps or empties? Did you adjust the final volumes? How did you avoid the absorbance by the culture dilutions? How can I distinguish between mycelium interference with light and absorbance? I suppose the culture is not homogeneous ¿what is the principle to determinate the mycelial growth by spectrophotometry? What characteristics of the spectrophotometer has to allow you to determine fungal growth? Furthermore, statical fungal cultures, sporulates on the interphase air/water which could be a problem. The other problem could be the secreted metabolites by the fungi. Lines 217-219. Did you observe a reverse correlation for the added volume from highest to lowest with the slight increase in fungal growth? It would be better if you show the 48-well microtiter plate representative photos. Line 261. I think the expression "data not shown" is not allowed in this journal. Please show the data. Lines 274-276. Please provide the quantification of the dual culture assays. I also would like to see a correlation analysis with both data. Lines 277-278. How many represent a large number for you? Considering this point what exactly you understand for HTP/HT? Line 415. How were the plates scanned and processed? Lines 423-424. Why the spore concentration varied for the different fungi? Please explain. Line 433-435. I am not sure if the determination of fungal growth spectrophotometrically is the best and accurate and reliable way to determine fungal growth. I would like to see the determination of fungal growth though time and the spectrophotometric data in a graphic with its respective correlation analysis.

Dear Author
The proposed work is very interesting and brings new approaches in the selection of beneficial microorganisms that can be used in the biological protection of plants against diseases caused by pathogenic fungi. Nevertheless, there is no empirical evidence supporting the advantages of the proposed methods over the current ones. The authors do not compare (numerically) the timeconsumption, costs and effectiveness of the available methods with those proposed. Just minimizing the experience to a 48-well microtiter plate does not justify its high throughput.The authors do not analyze the biotest preparation time, its duration and the time it takes to perform observations and measurements. The authors also do not indicate the costs generated by this approach. This knowledge could be used to compare the proposed approaches with existing methods -therefore some of the results presented in the manuscript are rather discussion and conclusions are not substantiated. I would like to point out that the resolution, visibility of some photos - Fig. 2 and Fig. S1 is very poor. The results cannot be properly assessed.
These and other comments are included in the PDF of the manuscript.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website. Large screens of bacterial strain collections to identify potential biocontrol agents are often time 12 consuming, costly, and fail to provide quantitative results. In this study, we present two quantitative 13 and high-throughput methods to assess the inhibitory capacity of bacterial biocontrol candidates 14 against fungal phytopathogens. One method measures the inhibitory effect of bacterial culture 15 supernatant components on the fungal growth, while the other accounts for direct interaction between 16 growing bacteria and the fungus by co-cultivating the two organisms. The antagonistic supernatant 17 method quantifies the culture components' antifungal activity by calculating the cumulative impact of Importance: Fungal phytopathogens are responsible for tremendous agricultural losses on annual 28 basis. While microbial biocontrol agents represent a promising solution to the problem, there is a 29 growing need for high-throughput methods to evaluate and quantify inhibitory properties of new 30 potential biocontrol agents for agricultural application. In this study, we present two high-throughput 31 and quantitative fungal inhibition methods that are suitable for commercial biocontrol screening. Streptomyces and the Bacillus genera are well known for their antifungal capacity and for the 59 production of a large variety of bioactive metabolites [15,[18][19][20][21][22][23]. Although, the inhibitory effect of 60 specific soil bacteria is well documented and recognized, there is a lack of quantitative and HTP 61 screening procedures to identify competent biocontrol agents. Consequently, many potential 62 biocontrol agents eventually fail to suppress plant diseases in field trials [24,25]. Classic antagonistic 63 screens assess the impact of the biocontrol candidates on the phytopathogen after co-inoculation on 64 solid media, which are referred to as dual-culture, plate confrontation or inhibition zone assays [24,26]. 65 Such methods account for numerous factors including nutrients or space competition, cell surface 66 components and the induced or constitutive secretion of volatile or soluble metabolites [20,[26][27][28]. 67 Other antagonistic assays evaluate the effect of individual inhibitory components on the 68 phytopathogens' growth like volatiles, polyketides, lipopeptides, siderophores and lytic enzymes 69 including, chitinases, glucanases, and proteases [20,26]. More complex antagonistic assays, such as leaf 70 disc or seedlings assays [29,30], investigate the tripartite interaction between biocontrol candidate, 71 phytopathogen and plant host, while non-antagonistic assays assess the importance of complementary 72 inhibitory mechanisms including niche colonization and priming of the plant immune response [26]. 73 Nevertheless, most screening systems are low throughput and provide only semi-quantitative 74 measurements of the inhibition potential against the fungus. Therefore, there is a need to develop 75 more efficient screening methods combining quantitative measurements of antimicrobial activity with 76 automation to increase speed and reduce resources required for the identification of good candidates. 77 Here, we describe two fungal inhibition methods to evaluate antifungal potency of potential biocontrol OD600 of each strain used in the study was correlated to viable cell counts (colony forming units (CFUs)). 104 A fungal spore suspension was prepared and mixed thoroughly to ensure a homogeneous spore 105 distribution. Then, the bacterial dilution series were co-inoculated with a fixed quantity of fungal spores 106 to determine the minimal inhibitory cell concentration (MICC) that abolishes fungal growth (Fig. 1B). 107 With this setup, a low MICC indicates a higher antifungal potency for a given bacterial strain. The assay 108 was prepared on appropriate agar medium for fungal cultivation in 48-well microtiter plates and 109 incubated 5 days at room temperature before assessing the fungal growth.  Table S1). The strains B. amyloliquefaciens BCF007 and B. velezensis BCF015 showed 145 the most potent inhibition properties, and even at the highest dilution step (corresponding to an initial 146 OD600 of 6.4*10 -6 , equivalent to 3-5 CFUs inoculated), the two strains were able to inhibit F. culmorum 147 growth. Determination of the minimal cell concentration that inhibits fungal growth even more 148 accurately would require to conduct the assay using serial dilutions at smaller dilution factors. 149 Nevertheless, the experimental setup is optimized for HTP screening of large strain collections. 150 B. subtilis BCF001 also displayed potent fungal inhibition properties, and abolished fungal growth up 151 to an OD600 of 3.2*10 -5 , corresponding to 19 CFU inoculated. B. paralicheniformis BCF009, however, 152 did not affect fungal growth even at the lowest dilution step, corresponding to an OD600 of 4*10 -3 , To generate visual and directly comparable plots of the inhibition results, we calculated a numerical 156 inhibition score that reflects the inhibitory capacity of each strain. In brief, the lowest cell concentration 157 that caused fungal growth inhibition was identified for each strain, and a numerical inhibition score was 158 calculated based on the natural logarithm to the MICC, by applying the empirical formula #1 (see 159 material and methods section) (Fig. 3A, Table S1). Plotting the inhibition scores clearly indicated that  Table S1. Interestingly, our inhibition assay proved applicable to these additional species of 178 plant pathogenic fungi. The initial spore concentration was the only parameter that required 179 adjustment when testing growth inhibition against the new fungal strains. 180 The previously described scoring system was applied to the results obtained for the three fungal species 181 (Fig. 3)  The inhibition scores can be found in Table S1 Table S2). Both filtration and antibiotic addition had generally no effect on the fungal inhibitory 266 potency, suggesting that bioactive metabolites remain active after both procedures (Fig. S5, Table S2).

268
Comparison of the two methods proposed in the present study and the dual-culture assay 269 The two proposed quantitative HTP methods were compared to the common dual-culture assay using 270 plates inoculated with fungus and Bacilli (Fig. 1A). In accordance with results from the two methods,   For more accurate fungal growth estimation, the OD600 was measured using a 5x5 well-scanning matrix. 435 Following the evaluation of fungal growth, the supernatants were collected and plated on LB with

Reviewer #1 (Comments for the Author):
The manuscript describes two quantitative and high-throughput methods to evaluate the inhibitory capacity of bacterial biocontrol candidates against fungal phytopathogens. The first described method uses growing bacteria cocultured with the fungus, whereas the second one shows potential to measure the inhibitory effect of bacterial culture supernatant components on the fungal growth. Since my view, the manuscript is well written and technically sounds, however, I have some major and minor concerns depicted below: Lines 1-2. Since my view, the title must reflect that the methods were designed for the screening of biocontrol bacterial strains.
 The authors agree with the reviewer and therefore the tittle has been modified to: Quantitative high-throughput screening methods designed for identification of bacterial biocontrol strains with antifungal properties Line 25 and subsequently. Please write down ".spp" in plane letter instead of italic.
 .spp is corrected to regular font. Thank you for commenting.  In the manuscript, we chose to demonstrate the functionality of the assays with a few selected strains for simplicity. Consequently, a paragraph has been added to the manuscript about the throughput of the assays (see Line 78 and 244).
Lines 100-101. Spores and mycelium do not have the same structure and composition of their cell walls, so, why not to include mycelium?
 We chose to use fungal spores to include the impact of Bacilli on fungal spore germination in the assessment, in addition to the impact on fungal mycelial growth. In addition, normalization of fungal spore inoculum is much easier than normalization of mycelium inoculum size, which is challenging. Using mycelial inoculum could decrease the reproducibility and accuracy of the assays  In order to clarify we have introduced a text change in line 100: Fungal spores rather than mycelium were used as initial inoculum in the assay to allow assessment of biocontrol agents impact on spore germination in addition to fungal growth.    The OD dilution series indicated next to figure 2B has been corrected. Thank you for noticing the discrepancy between the figure and the text.
 The CFU's and OD's have been corrected and is in accordance with figure 2 now. Thank you for noticing. Figure 3A. Above, you concluded that B. amyloliquefaciens and B. velezensis showed the most potent inhibition properties, and even at the highest dilution step against F. culmorum. However, B. subtilis showed similar results. It is necessary an statistical analysis.
 The significance of MICCs from each strain against each fungus was calculated and added to supplementary data: o A sentence was added in the text referring to the supplementary table with calculated MICC significance by comparison of the strain with students t-test (Table S2). o In addition, a sentence was added to explain that similarly potent strains can be distinguished by adjusted the spectrum of the dilution series. Zoom in on the effective MICC so to speak (see line 133 and line 166).
Lines 191-202. How did you determine the fungal growth by OD in the antagonistic supernatant method? What did you do to avoid clumps or empties? Did you adjust the final volumes? How did you avoid the absorbance by the culture dilutions? How can I distinguish between mycelium interference with light and absorbance? I suppose the culture is not homogeneous ¿what is the principle to determinate the mycelial growth by spectrophotometry? What characteristics of the spectrophotometer has to allow you to determine fungal growth? Furthermore, statical fungal cultures, sporulates on the interphase air/water which could be a problem. The other problem could be the secreted metabolites by the fungi.
Below we add a point-by-point answer to the reviewer's concerns: .  The fungal growth medium (potato dextrose broth) shares almost the same absorbance as the bacterial growth medium (Lysogeny Broth) and so the addition of supernatant dilutions should not affect the OD of the clear medium. The final volumes were however not adjusted, but a reverse correlation between supernatant addition and fungal growth was nevertheless observed.  During assay preparation, the fungal spore solution was mixed thoroughly by vortex to ensure a homogeneous distribution and avoid clumping of spores.  To account for empty space and clumps an area scanning protocol was used for measuring the fungal growth in each well (25 measurements/well), as we noticed that a single point measurement was not adequate. The measures were averaged within each well. The procedure is described in the main text for clarification and materials and methods (see lines 183 and 411).  An additional figure ( Figure S2) has been added to supplemental showing the OD measurements throughout a fungal growth curve.  The fungal growth was evaluated before the sporulation and pigment production stages were reached, avoiding that they could potentially interfere with the optical density measurements (within 2-3 days cultivation).
Lines 217-219. Did you observe a reverse correlation for the added volume from highest to lowest with the slight increase in fungal growth? It would be better if you show the 48-well microtiter plate representative photos.
 A reverse correlation was observed between the added volumes (from lowest to highest) and the fungal growth (progressively decreasing). In some cases, the largest added volume (80ul) resulted in a slight increase in fungal growth in respect to the immediate lower added volume. This is probably related to the positive effect that higher content of nutrients added have on the final fungal growth that slightly counteracts the inhibitory effect of bioactive metabolites. Adding larger volumes from this point on would not bring the fungal growth inhibition effect further. A sentence was added in the manuscript to explain this phenomenon (see line 195).  The fungal growth in liquid is less clear on pictures as the cultivation time (2-3 days) does not allow for development of arial hyphae or pigmentation as observed in the antagonistic co-cultivation assay that was incubated for a longer time (5 days) to allow distinction between the two species. We consider that the pictures of the liquid cultures would not be informative, contrary to the optical density values that clearly show the fungal growth decrease in response to the bacterial supernatants' addition.
Line 261. I think the expression "data not shown" is not allowed in this journal. Please show the data.
 The data that the reviewer requests correspond to a picture of an empty cultivation plate with selection for bacterial growth (LB agar with nystatin). We estimated that this picture would not more informative than the sentence. To avoid the term "data not shown" we have rephrased the sentence to indicate that there was absence of bacterial growth (line 221).
Lines 274-276. Please provide the quantification of the dual culture assays. I also would like to see a correlation analysis with both data.
 We consider dual-culture assays to be semi-quantitative methods at best. Factors like the moisture of the agar plates or even the distance between the inoculation points of the biocontrol agent and the pathogen would bias the relative inhibition and lack reliability. Therefore, although dual-culture assays are simple and allow for a fast evaluation of biocontrol agents potential, the authors did not attempt a direct comparison of the methods.  To respond to the reviewers request we have analyzed data from the dual-culture assay plates, calculated the corresponding inhibition scores and performed statistical analysis (see below).  Quantification of fungal growth inhibition by classical dual culture assay. A volume of 10 µl fungal spore solution was inoculated in the center of a PDA plate. F. culmorum, F. graminearum and B. cinerea were inoculated in final concentrations of 1.1*10 7 spores/ml, 1.25*10 5 spores/ml and 2*10 7 spores/ml, respectively. In a perimeter surrounding the central fungal inoculum, 10 µl overnight culture were inoculated of the respective Bacillus cultures adjusted to OD 600 0.2. The plates were sealed with 3M tape ([Millipore] 0.5 cm) and incubated at room temperature for 6 days. The fungal growth was evaluated in ImageJ (Fiji).   As commented previously, these assays were afterwards used for evaluation of antifungal potential of 20000+ strains within a short time frame. For instance, the antagonistic co-inoculation assay accommodates 250 strains per day when using a Hamilton liquid handling robot allowing screening of 800-1000 strains per week. In the manuscript, we aim to demonstrate the functionality of the assay with a few selected strains for simplicity.

Line 415. How were the plates scanned and processed?
Fungal inhibition by the antagonistic co-inoculation assay was evaluated by visual inspection of the plates and the plates were imaged by scanning the top of the plates using an Epson Perfection V800 Photo device. Line 388-389. Text was modified to clarify how plates were scanned and processed.
Lines 423-424. Why the spore concentration varied for the different fungi? Please explain.
 The fungal stocks may not have the same germination efficiency. Hence, the spore concentration was adjusted for each species to allow visualization of bacterial supernatant impact. In addition, different fungal species may be more or less susceptible to bacterial metabolites (enzymes, lipopeptides and polyketides). Therefore, the fungal spore concentration was adjusted, also to enable visualization of bacterial supernatant impact and allow comparison of strains' potency. For each fungus, it is necessary to optimize the initial spore concentration that allow visualization of the dynamic dilution range of bacterial cultures.
Line 433-435. I am not sure if the determination of fungal growth spectrophotometrically is the best and accurate and reliable way to determine fungal growth. I would like to see the determination of fungal growth though time and the spectrophotometric data in a graphic with its respective correlation analysis.
 As stated previously fungal growth curves were added to the supplemental materials (see Figure  S2).